Trinucleotide repeat sequences in transcripts of affected genes have been found in fragile X-syndrome ("Fra-X;" CGG repeats in the 5' untranslated region), muscular atrophy ("SBMA;" CAG repeat in the coding region), myotonic dystrophy ("DM;" CTG repeat in the 3' untranslated region), and Huntington's disease ("HD;" CAG repeat in the coding region). These repeats appear to be present in the normal gene as well, but the number of tandem trinucleotide repeats is increased in the disease state. Therefore, the disease-causing genetic defect is called an "expanded trinucleotide repeat" or a "trinucleotide repeat expansion". The extent or degree of trinucleotide repeat expansion associated with the disease state varies for different genetic diseases. In each disease, however, there appears to be a relatively consistent number of tandem repeats below which the patient has no symptoms, and above which disease symptoms begin to appear. In general, increasing severity of disease symptoms correlates with an increased degree of repeat expansion (i.e., a greater number of tandem trinucleotide repeats) once the maximum normal number is exceeded (Harley et al., 1992, Nature, 355:545-46; Buxton et al., 1992, Nature, 355:547; Aslanidis et al., 1992, Nature, 355:548; Brook et al., 1992, Cell, 68:799-808; Mahadevan, 1992 Science, 255:1253-56; Fu et al., 1992, Science, 255:1256-58; Tsilfidis et al., 1992, Nature Genetics, 1:192-95).
The mechanisms for trinucleotide repeat expansion are not known, but many of the genetic diseases associated with this phenomenon exhibit "anticipation". That is, the severity of symptoms increases in succeeding generations, suggesting that replication errors may contribute to the repeat expansion.
An example of such a disease is myotonic dystrophy, a human neuromuscular genetic disease inherited in an autosomal dominant fashion. The genetic defect has multisystem effects, including myotonia and weakness, cardiac conduction defects, cataracts, male baldness, hypersomnia, abnormal glucose response and male testicular atrophy as well as abnormalities in other systems. The clinical presentation of myotonic dystrophy is variable and has been well characterized (P. S. Harper, 1989, Myotonic Dystrophy, Saunders, London, and Philadelphia, 2nd ed.). While the genetic bases of the disease are not known, the trinucleotide repeat sequence (CTG).sub.n has been found in the 3' -untranslated region of myotonic-protein kinase (Mt-PK) mRNA. The severity of the disease may increase from one generation to the next (anticipation) and is related to expansion of the (CTG).sub.n repeat sequence.
Biochemical studies have not shown any mutated or defective protein associated with myotonic dystrophy, but defects in membrane structure and function have been found. There is also evidence of reduced phosphorylation of membrane proteins in red blood cells (Roses et al., 1973, PNAS, 70:1855) and sarcolemmal membranes from muscle biopsies of DM patients (Roses and Appel, 1974, Nature, 250:245). Fu et al., 1993, Science, 260:235-38 have shown that the amount of Mt-PK mRNA and the corresponding protein decreases with increased repeat expansion in the myotonic dystrophy patient. The regulatory role of protein kinase in development and the physiological modulation of channel proteins is also reduced in myotonic dystrophy patients (J. Wang et al, 1992, Nature, 359:739; J. W. West, 1991, Science, 254:866). Fu et al., supra, have suggested that the decrease in myotonic protein kinase contributes to the severity of the disease by disrupting signal transduction and amplification pathways. In contrast, another study has shown no difference in mRNA levels in myotonic dystrophy patients (Brook et al., 1992, Cell supra).
The CTG trinucleotide repeat sequence is polymorphic in the normal population and undergoes various degrees of expansion in myotonic dystrophy patients (Brook et al., 1992, Cell, 68:799). The average number of CTG repeats in normal cells is about 5 (48%)-27. DM patients have at least 50 copies, and up to several hundred copies. More severe cases are associated with higher number of repeats. One possible explanation for the expansion of the trinucleotide repeat may be errors in DNA replication during meiotic cell division or in the rapidly dividing cells of the early embryo. That is, replication of five trinucleotide repeat alleles may be stable, whereas duplication or triplication may occur when 27 repeat alleles are involved due to error in the DNA replication step from one generation to the next. The CTG trinucleotide repeat is transcribed from the gene and is located about 500 bp upstream of the poly(A) signal in the mRNA. The gene is expressed in many tissues of the myotonic dystrophy patient and encodes a protein (Mt-PK) having a strong homology with the protein kinase gene family. Normal Mt-PK protein is encoded by a gene having a genomic sequence of 11.5 kb. The gene contains 14 exons and has been mapped to chromosome 19. It is not known at the present time whether expansion of the trinucleotide repeat affects transcription, transport or function of the mRNA.
Many genes and RNAs contain sequences similar or identical to the trinucleotide repeats known to be expanded in genetic diseases. Probes and primers directed to the repeat sequence hybridize to these sequences, which are unrelated to the genetic disease of interest, creating smears on Northern and Southern blots or producing non-specific target amplification. For example, both the rRNA genes and the histone genes are GC rich and can be expected to hybridize to trinucleotide repeat probes. Probes to the trinucleotide repeat sequences have therefore previously been used only for hybridization to isolated nucleic acid segments, such as for screening cDNA libraries (Li et al., 1992, Am. J. Hum. Genet., 51:(4 Suppl.), A41; Riggins et al., 1992, Am. J. Hum. Genet., 51:(4 Suppl.), A41). Because a variety of short trinucleotide repeat sequences can be found all over the genome, previous methods for detection and analysis of repeat expansion in uncloned DNA have focused on the use of probes and primers which hybridize to unique sequences flanking or otherwise closely linked to the trinucleotide repeat of interest. This approach for specific analysis of repeat expansion in a gene of interest has been applied to diagnosis of Huntington's Disease (The Huntington's Disease Collaborative Research Group, 1993, Cell, 72:971-983; Goldberg et al., 1993, Human Molec. Genet., 2:635-636), X-linked spinal and bulbar muscular atrophy (SBMA) (Yamamoto et al., 1992, Biochem. Biophys. Res. Commun., 182:507-513) and to identify polymorphisms in cloned sequences containing trinucleotide repeats (Riggins et al., 1992, supra). Warner et al. 1993, Molec. Cell Probes, 7:235-239 have reported a polymerase chain reaction (PCR) assay for detection of the trinucleotide repeat associated with Huntington's disease. This PCR method employs one primer which spans the repeat and a GC rich region of the gene, but retains amplification specificity by directing the second primer to a unique flanking sequence. Repeat-specific oligonucleotides have also been used to detect expanded repeats in the genome by Repeat Expansion Detection (RED--Schalling et al., 1993, Nature Genet., 4:135-39). RED is similar to the ligase chain reaction in that repeat-specific oligonucleotides are cyclically hybridized to repeats in the genome, ligated and denatured. Only long repeats in the target DNA can serve as templates for adjacent annealing of multiple complementary oligonucleotides, reportedly eliminating detection of non-expanded trinucleotide repeats elsewhere in the genome. Several of these prior art methods have been applied to diagnosis of myotonic dystrophy. See, for example, Fu et al., 1992, Science, supra; Mahadevan et al., 1992, Science, supra; Hecht et al., 1993, Clin. Genet., 43:276-285; Brook et al., 1992, Cell, 68:799-808; WO 93/17104; WO 93/16196.
As discussed above, it is possible that the trinucleotide repeat sequence does not affect the transcription of the DM gene, but rather interferes with mRNA processing or transport to the cytoplasm. This would explain the reduction of Mt-PK mRNA and protein, and a similar abnormality has been reported in the double sex mutant of Drosophila, in which the repeat sequence binds to the protein involved in mRNA processing (Nagoshi, et al., 1990, Genes Dev., 4:89). Alternatively, the Mt-PK mRNA may be transported normally but may be dysfunctional in the cytoplasm. It is not possible, using prior art methods, to determine which of these mechanisms is operative.